There is a growing interest to use in vitro BBB cell assays in early safety assessment of compounds. By modifying a well-validated co-culture model of brain capillary endothelial and glial cells, developed by Dehouck et al. [Dehouck, M.P., Meresse, S., Delorme, P., Fruchart, J.C., Cecchelli, R., 1990. An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. Journal of Neurochemistry 54 (5), 1798-1801], it has been possible to develop a new in vitro BBB system suitable for high throughput screening (HTS). In addition, this new procedure substantially reduces the use of experimental animals and considerably facilitates the process of obtaining a functional in vitro BBB model. The model is ready to use after only 4 days of culture and then shows the typical expression and localization of tight junction proteins. The function of the P-glycoprotein and the transcriptional expression of other efflux transporters such as MRP 1, 4 and 5 have been demonstrated. In addition, the model produces a good in vitro/in vivo correlation for 10 compounds (R2=0.81). Furthermore, studies were undertaken within the European ACuteTox consortium with the objective to assess BBB toxicity and make risk assessments of potentially toxic compounds according to their predicted ability to reach the CNS compartment. These investigations demonstrated that the results produced in the HTS BBB model were similar to the standard co-culture model.